(1) Field of the Invention
The present invention relates to antibodies specific for phosphorylation sites and methods of screening drugs using the same antibodies. In more particular, the invention relates to novel antibodies that specifically recognize the phosphorylation site in the linker regions of Smad2 and Smad3, which are signal transduction molecules of the transforming growth factor-β (TGF-β) family. Further, the invention relates to methods of screening drugs that inhibit the phosphorylation in the linker regions of Smad2 and Smad3.
(2) Description of the Related Art
The TGF-β superfamily includes TGF-β, BMP, etc. and forms a multi-functional protein family having various physiological activities and cell regulatory functions, such as cell differentiation, growth inhibition and tissue repair, in living organisms. TGF-β is a representative cytokine of the superfamily and known to stimulate or inhibit the transcription activity of a target gene group by its signal transduction cascade.
TGF-β is known to be produced in various tissues such as platelet and act on various cells. TGF-β suppresses the growth of epithelial cells, such as hepatocyte, while promoting the accumulation of extracellular matrix (ECM) in mesenchyme cells such as fibloblast to repair tissue.
Activated Smad directly binds to the promoter region of ECM proteins, such as type I collagen and plasminogen activator inhibitor type I (PAI-1), and forms complexes with other transcription factors such as AP-I and ATF-2 to activate their transcription.
TGF-β receptors come in two types depending on the molecular weight: type I and type II and these are expressed in almost all the cells. The receptors each have serine/threonine kinase regions in the cells.
TGF-β firstly binds to its type II receptor and then to its type I receptor to form a complex. During the complex formation, the type II receptor phosphorylates the type I receptor, and then the type I receptor activated by the phosphorylation phosphorylates signal transducers called Smads which lie downstream of the TGF-β signaling system. As a result, the signals are transduced into cells (Heldin et al., Nature 390: 465-471, 1997). Smads activated by the receptor are translocated into the nucleus and act as transcription factors of target genes.
Members of the Smad protein family were identified based on the homology with Mad (mothers against Dpp) obtained by screening of genes that modified the signals of Dpp (decapentaplegic), which was a BMP homologue of Drosophila. On the other hand, genes, sma-2, -3 and -4, were obtained, which showed the same phenotype as the abnormality of gene daf-4 of receptor DAF-4, a receptor of BMP-like factor of C.elegance. Since both Sma proteins encoded by these genes, and Mad transduce the signals of the TGF-β superfamily and have a high degree of amino acid homology to one another, they are called Smad. Generally, Smad proteins are composed of about 500 amino acid residues and eight subtypes, Smad1 to Smad8, have been identified.
Smad proteins are classified into 3 types depending on their structure and function: R-Smads (receptor-regulated Smads) that are activated by type I receptor and transduce signals specific for the TGF-β and BMP signaling system, respectively; Co-Smads (common-mediator Smads) that are commonly used in all the signaling system and form complexes with R-Smads; and I-Smads (inhibitory Smads) that act in an inhibitory manner on the R-Smads and Co-Smads. As R-Smads, Smads1 to 3, 5 and 8 are known, as Co-Smads, Smad 4, and as I-Smads, Smads 6 and 7 are known.
Generally, Smads have two domains called MH1 (Mad homology 1) domain in the N-terminal region and MH2 domain in the C-terminal region, which are highly conserved among the Smad family. The MH1 domain and the MH2 domain are linked together in the region called “linker region”.
It is known that in the linker regions of Smad1, Smad2 and Smad3, Ser residues are phosphorylated by Erk (Kretzschmar et al., Nature 389: 618-622, 1997; Kretzschmar et al., Genes Dev. 13: 804-816, 1999). Likewise, Ser240 and Ser260 in the linker region of Smad2 are phosphorylated by CamKII just as is Ser110 at the MH1 domain.
At the C-terminus of R-Smads there is an amino acid sequence, Ser-Ser-X-Ser (SSXS motif), and the two Sers proximate to the C-terminus are phosphrylated by TGF-β type I receptor.
Smads play a role specific for the signaling of TGF-β family members, and Smad2 and Smad3 are specific for TGF-β signaling (Heldin et al. supra). Activated Smad2 and Smad3 interact with Smad 4, which is a Co-Smad, and are translocated into the nucleus to activate target genes. TGF-β signaling activates Smad2 and Smad3 while allowing TGF-β signaling to be balanced by Smad6 and Smad7 as I-Smads.